Bifidobacterium longum and functional GI disorders

ABSTRACT

The present invention generally relates to the field of probiotic bacteria. In particular it relates to methods for treating functional GI disorders comprising administering Bifidobacterium longum, such as Bifidobacterium longum ATCC BAA-999.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a National Stage of International ApplicationNo. PCT/EP2010/062320, filed on Aug. 24, 2010, which claims priority toEuropean Patent Application No. 09168590.9, filed on Aug. 25, 2009, theentire contents of which are being incorporated herein by reference.

The present invention generally relates to the field of probioticbacteria. In particular it relates to Bifidobacterium longum NCC3001(ATCC BAA-999) subsequently referred to as Bifidobacterium longum ATCCBAA-999, and its use in ingestible compositions. The compositiondescribed in the present invention may be used to treat or preventfunctional gastro-intestinal (GI) disorders.

Functional GI disorders (FGIDs) are a group of disorders includingIrritable bowel syndrome (IBS) and Functional dyspepsia that are chronicconditions associated with high morbidity characterized by abdominaldiscomfort or pain, bloating and in the case of IBS altered bowel habits(diarrhoea and/or constipation).

The prevalence of FGIDs in the general population is relatively highranging from 15%-30% having a substantial economic burden. It has beensuggested that the annual direct costs for IBS alone are now around$US41 billion in the 8 most industrialised countries with considerableadditional indirect costs (eg absenteeism from work etc). Currenttreatments for FGIDs demonstrate at best marginal efficacy which hasbeen historically related to poor understanding of the diseasepathogenesis.

The precise pathophysiology of IBS remains to be elucidated.

Recent studies have described mucosal inflammation and alterations inintestinal microflora in IBS patients and a disease correlation withintestinal infections.

The fact that some probiotics exhibit anti-bacterial, anti-viral andanti-inflammatory properties and that they can restore the intestinalmicrobiota balance, suggested that they may become suitable therapeuticagents for IBS.

To date several studies on the effect of different probiotics on IBSsubjects have been published. These studies suggest that probiotic usemay be associated with improvement in IBS symptoms but also that not allprobiotics have equal efficacy in IBS.

Several meta-analysises performed recently concluded that there isinadequate data to comment on the efficacy of other probiotics (Lynne VMcFarland, et al., World J Gastroenterol 2008 May 7; 14(17): 2650-2661;Nourieh Hoveyda, et al., BMC Gastroenterology 2009, 9:15).

Consequently, it was the object of the present invention to improve thestate of the art and in particular to provide the art with a compositioncomprising an alternative bacterial strain that is effective, readilyavailable, low priced and safe to administer without unwanted sideeffects which can be used to treat or prevent functional GI disorders.

The present inventors have addressed this need. They were surprised tosee that they could achieve this object by the subject matter of theindependent claims. The dependant claims further develop the idea of thepresent invention.

It was found that the effectiveness in treating and/or preventingfunctional GI disorders depends on the bacterial genus, species andstrain used.

Consequently, one embodiment of the present invention is a compositioncomprising Bifidobacterium longum ATCC BAA-999, and/or its fermentedgrowth medium for use in the treatment and/or prevention of functionalGI disorders.

The present invention also relates to the use of Bifidobacterium longumATCC BAA-999 for the preparation of a composition to treat and/orprevent functional GI disorders.

Bifidobacterium longum ATCC BAA-999 was one strain of the speciesBifidobacterium longum that was found to be in particular effective inachieving the object of the present invention.

Advantageously, Bifidobacterium longum ATCC BAA-999 is commerciallyavailable and was already tested and found to be acceptable for additionto food products, for example.

Bifidobacterium longum ATCC BAA-999 (BL999) may be obtained commerciallyfrom specialist suppliers, for example from Morinaga Milk Industry Co.Ltd. of Japan under the trade mark BB536.

Bifidobacterium longum ATCC BAA-999 (BL999) may be cultured according toany suitable method. It may be added to products in a freeze-dried orspray-dried form, for example.

The term “Bifidobacterium longum ATCC BAA-999 (BL999)” is meant toinclude the bacterium, parts of the bacterium and/or a growth mediumfermented by the bacterium.

The composition may be any composition, but is preferably a compositionto be administered orally, enterally or rectally.

For example, the composition may be an edible composition.

“Edible” means a material that is approved for human or animalconsumption.

Typically, the composition may be selected from the group consisting ofa food composition, a pet food composition, a dietary supplement, anutraceutical, a nutritional formula, a drink, and/or a medicalcomposition.

If the composition of the present invention is a food composition, thishas the advantage that such a composition can be distributed inpharmacies, drug stores, but also in normal supermarkets, where thecompositions are easily available to everybody.

The generally pleasant taste of food compositions will furthercontribute to the acceptance of the product. In particular smallchildren or pets are much more likely to readily consume compositionswith a taste that is generally liked.

Examples of food products that are applicable to the present inventionare yoghurts, milk, flavoured milk, ice cream, ready to eat desserts,powders for re-constitution with, e.g., milk or water, chocolate milkdrinks, malt drinks, ready-to-eat dishes, instant dishes or drinks forhumans or food compositions representing a complete or a partial dietintended for pets or livestock.

Consequently, in one embodiment the composition according to the presentinvention is a food product intended for humans, pets or livestock.

The composition may be intended for animals selected from the groupconsisting of dogs, cats, pigs, cattle, horses, goats, sheep or poultry.

In a preferred embodiment, the composition is a food product intendedfor adult species, in particular human adults.

The composition of the present invention may further contain protectivehydrocolloids (such as gums, proteins, modified starches), binders, filmforming agents, encapsulating agents/materials, wall/shell materials,matrix compounds, coatings, emulsifiers, surface active agents,solubilizing agents (oils, fats, waxes, lecithins etc.), adsorbents,carriers, fillers, co-compounds, dispersing agents, wetting agents,processing aids (solvents), flowing agents, taste masking agents,weighting agents, jellifying agents, gel forming agents, antioxidantsand antimicrobials. The composition may also contain conventionalpharmaceutical additives and adjuvants, excipients and diluents,including, but not limited to, water, gelatine of any origin, vegetablegums, ligninsulfonate, talc, sugars, starch, gum arabic, vegetable oils,polyalkylene glycols, flavouring agents, preservatives, stabilizers,emulsifying agents, buffers, lubricants, colorants, wetting agents,fillers, and the like. In all cases, such further components will beselected having regard to their suitability for the intended recipient.

The composition may be a nutritionally complete formula.

The composition according to the invention may comprise a source ofprotein.

Any suitable dietary protein may be used, for example animal proteins(such as milk proteins, meat proteins and egg proteins); vegetableproteins (such as soy protein, wheat protein, rice protein, and peaprotein); mixtures of free amino acids; or combinations thereof. Milkproteins such as casein and whey, and soy proteins are particularlypreferred.

The proteins may be intact or hydrolysed or a mixture of intact andhydrolysed proteins. It may be desirable to supply partially hydrolysedproteins (degree of hydrolysis between 2 and 20%), for example for humansubjects and/or animals at risk of developing cows' milk allergy.

Furthermore, pre-hydrolysed protein sources are generally easierdigested and absorbed by an impaired gastro-intestinal tract.

If hydrolysed proteins are required, the hydrolysis process may becarried out as desired and as is known in the art. It may be desirableto supply partially hydrolysed proteins (degree of hydrolysis between 2and 20%).

For example, a whey protein hydrolysate may be prepared by enzymaticallyhydrolysing the whey fraction in one or more steps. If the whey fractionused as the starting material is substantially lactose free, it is foundthat the protein suffers much less lysine blockage during the hydrolysisprocess. This enables the extent of lysine blockage to be reduced fromabout 15% by weight of total lysine to less than about 10% by weight oflysine; for example about 7% by weight of lysine which greatly improvesthe nutritional quality of the protein source.

The composition may also contain a source of carbohydrates and a sourceof fat.

If the composition includes a fat source, the fat source preferablyprovides 5% to 40% of the energy of the composition; for example 20% to30% of the energy. A suitable fat profile may be obtained using a blendof canola oil, corn oil and high-oleic acid sunflower oil.

A source of carbohydrate may be added to the composition.

The source of carbohydrates preferably provides 40% to 80% of the energyof the composition. Any suitable carbohydrate may be used, for examplesucrose, lactose, glucose, fructose, corn syrup solids, maltodextrins,and mixtures thereof. Dietary fibre may also be added if desired.Dietary fibre passes through the small intestine undigested by enzymesand functions as a natural bulking agent and laxative. Dietary fibre maybe soluble or insoluble and in general a blend of the two types ispreferred. Suitable sources of dietary fibre include soy, pea, oat,pectin, guar gum, partially hydrolysed guar gum, gum Arabic,fructooligosaccharides, acidic oligosaccharides,galacto-oligosaccharides, sialyl-lactose and oligosaccharides derivedfrom animal milks. A preferred fibre blend is a mixture of inulin withshorter chain fructo-oligosaccharides. Preferably, if fibre is present,the fibre content is between 2 and 40 g/l of the composition asconsumed, more preferably between 4 and 10 g/l.

The composition may also contain minerals and micronutrients such astrace elements and vitamins in accordance with the recommendations ofGovernment bodies such as the USRDA. For example, the composition maycontain per daily dose one or more of the following micronutrients inthe ranges given:—300 to 500 mg calcium, 50 to 100 mg magnesium, 150 to250 mg phosphorus, 5 to 20 mg iron, 1 to 7 mg zinc, 0.1 to 0.3 mgcopper, 50 to 200 μg iodine, 5 to 15 μg selenium, 1000 to 3000 μg betacarotene, 10 to 80 mg Vitamin C, 1 to 2 mg Vitamin B1, 0.5 to 1.5 mgVitamin B6, 0.5 to 2 mg Vitamin B2, 5 to 18 mg niacin, 0.5 to 2.0 μgVitamin B12, 100 to 800 μg folic acid, 30 to 70 μg biotin, 1 to 5 μgVitamin D, 3 to 10 μg Vitamin E.

One or more food grade emulsifiers may be incorporated into thecomposition if desired; for example diacetyl tartaric acid esters ofmono- and di-glycerides, lecithin and mono- and di-glycerides. Similarlysuitable salts and stabilisers may be included.

The composition may be orally and/or enterally administrable; forexample in the form of a powder for re-constitution with milk or water.

According to one preferred embodiment of the present invention thecomposition comprises at least one other kind of other food grademicro-organism.

“Food grade” micro-organisms are micro-organisms that are safe for usein food.

The food grade micro-organisms are preferably food-grade bacteria orfood-grade yeast. The food grade bacteria may be selected from the groupconsisting of lactic acid bacteria, bifidobacteria, propionibacteria ormixtures thereof. As food grade yeast for example Saccharomycescerevisiae and/or Saccharomyces boulardii can be used.

The food grade bacteria may be probiotic bacteria.

“Probiotic” means microbial cell preparations or components of microbialcells with a beneficial effect on the health or well-being of the host.(Salminen S, Ouwehand A. Benno Y. et al “Probiotics: how should they bedefined” Trends Food Sci. Technol. 1999:10 107-10).

Probiotic bacteria are preferably selected from the group consisting oflactic acid bacteria, bifidobacteria, propionibacteria or mixturesthereof. Probiotic bacteria may be any lactic acid bacteria orbifidobacteria with established probiotic characteristics. For examplethey may be also capable of promoting the development of a bifidogenicintestinal microbiota.

Suitable probiotics may be selected from the group consisting ofBifidobacterium, Lactobacillus, Streptococcus and Saccharomyces ormixtures thereof, in particular selected from the group consisting ofBifidobacterium longum, Bifidobacterium lactis, Lactobacillusacidophilus, Lactobacillus rhamnosus, Lactobacillus paracasei,Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillussalivarius, Enterococcus faecium, Saccharomyces boulardii andLactobacillus reuteri or mixtures thereof, preferably selected from thegroup consisting of Lactobacillus johnsonii (NCC533; CNCM 1-1225),Bifidobacterium longum (NCC490; CNCM 1-2170), Bifidobacterium longum(NCC2705; CNCM 1-2618), Bifidobacterium lactis (2818; CNCM 1-3446),Lactobacillus paracasei (NCC2461; CNCM 1-2116), Lactobacillus rhamnosusGG (ATCC53103), Lactobacillus rhamnosus (NCC4007; CGMCC 1.3724),Enterococcus faecium SF 68 (NCIMB10415), and mixtures thereof.

In a preferred embodiment of the present invention the compositionfurther contains at least one prebiotic. “Prebiotic” means foodsubstances intended to promote the growth of probiotic bacteria in theintestines.

Prebiotics can thus promote the growth of certain food grade bacteria,in particular of probiotic bacteria, in the intestines and can henceenhance the effect of Bifidobacterium longum ATCC BAA-999. Furthermore,several prebiotics have a positive influence on, e.g., digestion.

Preferably the prebiotic may be selected from the group consisting ofoligosaccharides and optionally contain fructose, galactose, mannose,soy and/or inulin; dietary fibers; or mixtures thereof.

The Bifidobacterium longum ATCC BAA-999, may be used, both, as livingbacterium as well as inactivated non-replicating bacterial species.

“Non-replicating” means that no viable cells and/or colony forming unitscan be detected by classical plating methods. Such classical platingmethods are summarized in the microbiology book: James Monroe Jay,Martin J. Loessner, David A. Golden. 2005. Modem food microbiology. 7thedition, Springer Science, New York, N.Y. 790 p. Typically, the absenceof viable cells can be shown as follows: no visible colony on agarplates or no turbidity in liquid growth medium after inoculation withdifferent concentrations of bacterial preparations (‘non replicating’samples) and incubation under appropriate conditions (aerobic and/oranaerobic atmosphere for at least 24 h).

It is preferred that at least a part of the Bifidobacterium longum ATCCBAA-999, are alive in the composition and preferably arrive alive in theintestine. This way they can persist in the intestine and may increasetheir effectiveness by multiplication. They may also be effective byinteracting with the commensal bacteria and/or the host.

For special sterile food products or medicaments, for example, it mightbe preferable that Bifidobacterium longum ATCC BAA-999 is present in anon-replicating form in the composition. Hence, in one embodiment of thepresent invention at least a part of the Bifidobacterium longum ATCCBAA-999, are non-replicating in the composition.

In therapeutic applications, compositions are administered in an amountsufficient to at least partially cure or arrest the symptoms of thedisease and its complications. An amount adequate to accomplish this isdefined as “a therapeutically effective dose”. Amounts effective forthis purpose will depend on a number of factors known to those of skillin the art such as the severity of the disease and the weight andgeneral state of the patient.

In prophylactic applications, compositions according to the inventionare administered to a patient susceptible to or otherwise at risk of aparticular disease in an amount that is sufficient to at least partiallyreduce the risk of developing a disease. Such an amount is defined to be“a prophylactic effective dose”. Again, the precise amounts depend on anumber of patient specific factors such as the patient's state of healthand weight.

Generally, Bifidobacterium longum ATCC BAA-999 will be administered in atherapeutically effective dose and/or in a prophylactic effective dose.

If Bifidobacterium longum ATCC BAA-999 is present in a viable form, itis theoretically effective in any concentration considering the factthat these bacteria can colonize the gut and multiply.

For the composition of the present invention it is generally preferredthat a daily dose of the composition comprises between 10⁴ and 10¹² cfu(colony forming units) of Bifidobacterium longum ATCC BAA-999. Aparticular suitable daily dose of Bifidobacterium longum ATCC BAA-999,is from 10⁴ to 10¹¹ cfu, more preferably from 10⁴ to 10¹⁰ cfu.

The composition of the present invention may also comprise between 10²and 10¹⁰ cfu, preferably 10² to 10⁹ colony forming units, morepreferably from 10² to 10⁸ cfu of Bifidobacterium longum ATCC BAA-999,per gram dry weight of the composition.

In the case of inactivated and/or non-replicating Bifidobacterium longumATCC BAA-999, it is generally preferred that the composition of thepresent invention comprises between 10² and 10¹⁰ non-replicating cellsof Bifidobacterium longum ATCC BAA-999, per gram of the dry weight ofthe composition. A particular suitable dose of Bifidobacterium longumATCC BAA-999, is from 10³ and 10⁸ non-replicating cells, more preferablyfrom 10⁵ and 10⁸ non-replicating cells per gram of the dry weight of thecomposition.

Obviously, non-replicating micro-organisms do not form colonies,consequently, the term cells is to be understood as the amount of nonreplicating micro-organisms that is obtained from the specified amountof replicating bacterial cells. This includes micro-organisms that areinactivated, non viable or dead or present as fragments such as DNA orcell wall materials.

The composition of the present invention may be provided in powder formhaving a water activity smaller than 0.2, for example in the range of0.19-0.05, preferably smaller than 0.15.

The composition may be a shelf stable powder. The low water activityprovides this shelf stability and ensures that probiotic micro-organism,e.g., Bifidobacterium longum ATCC BAA-999, will remain viable even afterlong storage times. Water activity or a_(w) is a measurement of theenergy status of the water in a system. It is defined as the vaporpressure of water divided by that of pure water at the same temperature;therefore, pure distilled water has a water activity of exactly one.

Additionally or alternatively, the probiotic micro-organismBifidobacterium longum ATCC BAA-999 may be provided in an encapsulatedform.

It has been found that encapsulation of the bacteria has therapeuticaland technical advantages. Encapsulation increases the survival of thebacteria and thus the number of live bacteria which arrive in theintestine. Furthermore, the bacteria are gradually released allowing aprolonged action of the bacteria on the health of the subject. Bacteriamay be micro-encapsulated, for example as described by FR2443247(Societe des Produits Nestle), incorporated herein by reference.Briefly, the bacteria may be freeze or spray dried and incorporated intoa gel.

The present inventors were in particular surprised to find that thecomposition of the present invention can successfully be used tosignificantly increase hippocampal BDNF expression.

BDNF (Brain-derived neurotrophic factor) is a growth factor from aunique family of polypeptide growth factors. BDNF and other neurotrophicfactors, e.g., NGF (nerve growth factor), NT-3 (neurotrophin-3), andNT-4 (neurotrophin-4) are essential for the health and well-being of thenervous system.

This effect might possibly explain the observed effect againstfunctional bowel disorders.

The term “functional GI disorder” refers to a group of bowel disorderswhich are characterised by chronic abdominal complaints without astructural or biochemical cause that could explain symptoms.

Functional GI disorders are well known to those of skill in the art andare fore example described and defined as functional gastrointestinaldisorders with symptoms attributable to the middle or lowergastrointestinal tract by Longstreth et al., in GASTROENTEROLOGY 2006;130:1480-1491, for example.

The functional GI disorder may be selected from the group consisting ofirritable bowel syndrome; functional dyspepsia; functional constipation,functional diarrhoea; functional abdominal pain; functional bloating,Epigastric Pain Syndrome, Postprandial Distress Syndrome or combinationsthereof.

Functional GI disorders that may be treated or prevented by the subjectmatter of the present invention comprise anxiety linked functional GIdisorders, for example.

Anxiety is a psychological and physiological state which results in anunpleasant feeling that is typically associated with uneasiness, fear,or worry. Anxiety is for example a normal reaction to stress. It mayhelp a person to deal with a difficult situation at work or at school,but—when excessive—anxiety disorders result, one group of which areanxiety linked functional GI disorders.

The inventors—without wishing to be bound by theory—presently believethat the underlying mechanism by which the compositions of the presentinvention are effective is related to the modulation of thebidirectional microbial-gut-brain axis, possibly significantlyassociated with psychological factors.

It is clear to those skilled in the art that any features described inthis specification can be combined freely without departing from thescope of the present invention as disclosed. In particular, all featuresdescribed for the composition of the present invention are applicable tothe use of the present invention and vice versa.

Further features and advantages of the present invention result from thefollowing Examples and Figures:

FIG. 1 shows the result of a dark box/bright box test: total time spentin bright box, latency to re-enter bright box and latency to step downin mice infected with Trichuris muris (Tm). Tm-medium and Tm-B. longumare Tm infected mice treated with fresh medium (negative control) andBifidobacterium longum ATCC BAA-999, respectively; a Tm group treatedwith L. rhamnosus (L. rh) strain is shown for comparison.

FIG. 2 shows the results of the in situ hybridization in the brainhippocampal region of mice infected with a Trichuris muris (Tm). Tm-B.longum is Tm infected mice treated with Bifidobacterium longum ATCCBAA-999; a Tm group of mice treated with L. rhamnosus strain is shownfor comparison. Quantification of 35S signals was performed byautoradiography and image analysis (right upper panel).

FIG. 3 shows the results obtained on colonic inflammation measured bymyeloperoxidase activity assay (left panel) and mononuclear cellinfiltration (right panel) in mice infected with Trichuris muris (Tm).Tm-medium and Tm-B. longum are Tm infected mice treated with freshmedium (negative control) and Bifidobacterium longum ATCC BAA-999; a Tmgroup of mice treated with L. rhamnosus strain is shown for comparison.

EXAMPLES

Material and Methods

Bacterial Culture Conditions:

Probiotics (Bifidobacterium longum ATCC BAA-999 and L. rhamnosus NCC4007for comparison) were grown under anaerobic conditions inMan-Rogosa-Sharpe (MRS, BioMerieux) broth (bifidobacteria with 0.5%cysteine). After 24 h at 37° C., the number of bacteria was estimated bymeasuring the optical density at 600 nm (1 OD600=10⁸ bacteria/mL).Bacterial cells were pelleted by centrifugation at 5000×g for 15 min at4° C. and further resuspended at a concentration of 10¹⁰/mL in theirspent culture medium. Aliquots of 1 mL were kept frozen until use.

Animals:

Male BALB/c or AKR mice (Harlan, Canada) were purchases at the age of6-8 wks and housed in a conventional specific pathogen free Unit atMcMaster University Central Animal Facility. All experiments wereconducted with approval from the McMaster University Animal CareCommittee.

Design:

Chronic T. muris Infection:

Male AKR mice were gavaged with T. muris (300 eggs/mouse) (n=26) or withplacebo (n=9). Infected mice were then gavaged daily with L. rhamnosus,B. longum or fresh MRS from day 30 for 10 days. Uninfected mice weregavaged with fresh MRS on a daily basis from day 30 to day 40. At theend of probiotic or placebo administration, the mice underwent darkbox/bright box and step-down tests. The mice were sacrificed thereafterand tissue samples were obtained. Colon samples were fixed in formalinfor histological analysis or were snap frozen for MPO determination.Brains were snap frozen in liquid nitrogen and stored for in situhybridization.

Behavior Testing:

Dark Box/Bright Box:

Anxiety behavior was assessed individually in mice using dark box/brightbox as described in the literature. Briefly, each mouse was placed inthe center of illuminated bright box (30×30 cm) connected by an opening(10×3 cm) to smaller dark box (30×15 cm). The locomotor behavior of eachmouse in the bright box was recorded for 10 min by a digital videocamera and stored in a computer for an off-line analysis. Severalparameters were assessed by a blinded observer including total timespent in bright box, latency to re-enter bright box (time spent in darkbox after first entry), and number of crossovers (number of crossingfrom dark box to bright box).

Step-Down Test:

Anxiety behavior was assessed using step down test as described in theliterature. Briefly, each mouse was placed in the center of an elevatedplatform (7.5 cm in diameter, 3 cm high) positioned in the middle of ablack floor. Latency to step down from the pedestal was measured by astop-watch; maximum duration of the test was 5 min.

Histology:

Colon samples were fixed in 10% formalin and then stained withhematoxylin-eosin. The slides were examined under light microscopy tograde for acute and chronic inflammatory infiltrate.

Myeloperoxidase Activity Assay:

In order to assess acute intestinal inflammation, myeloperoxidaseactivity (MPO) assay was performed on frozen tissues as describedpreviously. MPO activity is expressed in units per mg of tissue, whereone unit of MPO is defined as a quantity of the enzyme able to convert 1μmol of hydrogen peroxide to water in 1 minute at room temperature.

In Situ Hybridization in the CNS:

Levels of BDNF in hippocampus and CRH in hypothalamus (paraventricularnucleus) were assessed by in situ hybridizations using 35S-labeled RNAprobes on frozen brain sections as described previously (Whitfield etal., 1990; Foster et al., 2002). Briefly, brains were removed andrapidly frozen by immersion in 2-methylbutane at −60° C., and stored at−70° C. Cryostat-cut 12-pin-thick coronal sections were thaw-mountedonto gelatine-coated slides, dried, and stored at −35° C. Tissuesections were fixed with 4% formaldehyde, acetylated with 0.25% aceticanhydride in 0.1M triethanolamine-HCl, pH 8.0, dehydrated, anddelipidated with chloroform. Antisense BDNF ribonucleotide probe (giftof Dr. J. Lauterborn and Dr. C. Gall, Univeristy of California Irvine)and anti-sense CRH ribonucleotide probe (gift of Dr. James Herman,University of Cincinnati) was transcribed from linearized plasmid usingthe Riboprobe System (Promega Biotech, Burlington, ON) with α-35S-UTP(specific activity>1000 Ci/mmol; Perkin-Elmer, Boston, Mass.) and T3 andT7 polymerases respectively. Radiolabelled probes were diluted in ahybridization buffer (0.6M NaCl, 10 mM Tris pH 8.0, 1 mM EDTA pH 8.0,10% Dextran sulfate, 0.01% sheared salmon sperm DNA, 0.05% total yeastRNA, type XI, 0.01% yeast tRNA, 1×Denhardt's solution) and applied tobrain sections (approximately 500,000 CPM/section). Slides wereincubated overnight at 55° C. in a humidified chamber. To reducenonspecific binding of the probe, slides were washed in 20 μg/ml RNasesolution for 30 min at room temperature, followed by 1 h each in 2×SSCat 50° C., 0.2×SSC at 55° and 60° C. Slides were dehydrated andair-dried for autoradiography. Slides and 14 C plastic standards wereplaced in x ray cassettes, apposed to film (BioMax MR; Eastman Kodak,Rochester, N.Y.) for 5 days and developed (Kodak Medical X-RayProcessor). Autoradiographic film images of brain sections and standardswere digitized with a solid-state camera with a 60 mm camera lensmanufactured by Nikon and using QCapture software (Qicam; QuorumTechnologies Inc., Guelph, ON) and a Macintosh computer-based imageanalysis system with Image software. Light transmittance through thefilm was measured by outlining the structure on the monitor. For BDNFmRNA, transmittance was converted to radioactivity levels using theRodbard curve applied to the standards. For CRH mRNA, the density slicefeature was utilized to measure both the light transmittance and thearea of mRNA signal. The calculated DPM were then multiplied by area toproduce a measurement of integrated density. Illustrations were madedirectly from the captured images.

Statistical Analysis:

Data are presented as mean±standard deviation or medians withinterquartile ranges as appropriate. Data was analysed using eithertwo-way ANOVA, test or non-paired t-test as appropriate. A p value of<0.05 was considered as statistically significant.

Results:

Mice chronically infected with the parasite Trichuris muris showed anincrease in anxiety-like behaviour in two behavioural tests: 1) In thedark box/bright box test, infected animals showed a decrease in the timespent in the bright box and an increase of the latency to re-enter thebright box; 2) In the step down test, the infection increased thelatency to step down from the pedestal (FIG. 1). Treatment withBifidobacterium longum ATCC BAA-999 but not with L. rhamnosus NCC4007induced a reduction of anxiety-like behaviour towards normality. Theeffect on behaviour was correlated with a normalization of Trichurismuris-mediated decrease in BDNF levels in the hippocampus by B. longumonly (FIG. 2). In contrast, treatment with B. longum as well as with L.rhamnosus resulted in a reduction of myeloperoxidase activity andmononuclear infiltration previously induced by Trichuris muris infection(FIG. 3) indicating that the normalization of behaviour was independentof the anti-inflammatory effect of the bacteria.

The invention claimed is:
 1. A method for the treatment of irritablebowel syndrome and anxiety, the method comprising: administering to apatient suffering from the same a composition comprising Bifidobacteriumlongum ATCC BAA-999, wherein the composition is selected from the groupconsisting of a dietary supplement, a nutraceutical, a drink, and amedical composition.
 2. The method of claim 1, wherein the compositioncomprises at least one other type of food grade bacteria.
 3. The methodof claim 1, wherein the composition comprises at least one prebiotic. 4.The method of claim 3, wherein the prebiotic is selected from the groupconsisting of oligosaccharides, dietary fibers, and mixtures thereof. 5.The method of claim 1, wherein at least 5% of Bifidobacterium longumATCC BAA-999 cells are viable in the composition.
 6. The method of claim1, wherein at least 80% of Bifidobacterium longum ATCC BAA-999 cells arenon-replicating in the composition.
 7. The method of claim 1, whereinthe composition comprises between 10⁴ and 10¹⁰ cells of Bifidobacteriumlongum ATCC BAA-999 per g dry weight of the composition.
 8. The methodof claim 1, wherein the composition comprises between 10² and 10⁸ cellsof Bifidobacterium longum ATCC BAA-999 per g dry weight of thecomposition.
 9. The method of claim 1, wherein the composition comprisesa protein source comprising hydrolyzed whey protein.
 10. The method ofclaim 1, wherein the composition is in powder form having a wateractivity of less than 0.2.
 11. The method of claim 2, wherein the atleast one other type of food grade bacteria is selected from the groupconsisting of lactic acid bacteria, propionibacteria and mixturesthereof.